The electrophoretic mobility shift assay (EMSA) is a method for the study of specific DNA-protein interactions in vitro. The pregnane X receptor (PRX) is a key xenobiotic sensor that regulates the expression of drug-metabolizing enzymes and many other genes. Radiolabeled P-32-DNA-probes had been used in studies of PXR-DNA interactions. There is an increasing need for nonradioactive assays, due to the health, safety and environmental issues. In the current study, we present a protocol for the nonradioactive electrophoretic mobility shift assay, allowing studying interactions between human PXR with promoter DNA sequences.